Recent findings in the animal model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis, implicate a novel CD4+ T-cell subset (TH17), characterized by the secretion of interleukin-17 (IL-17), in disease pathogenesis. To elucidate its role in MS, brain tissues from patients with MS were compared to controls. We detected expression of IL-17 mRNA (by in situ hybridization) and protein (by immunohistochemistry) in perivascular lymphocytes as well as in astrocytes and oligodendrocytes located in the active areas of MS lesions. Further, we found a significant increase in the number of IL-17+ T cells in active rather than inactive areas of MS lesions. Specifically, double immunofluorescence showed that IL-17 immunoreactivity was detected in 79% of T cells in acute lesions, 73% in active areas of chronic active lesions, but in only 17% of those in inactive lesions and 7% in lymph node control tissue. CD8+, as well as CD4+, T cells were equally immunostained for IL-17 in MS tissues. Interestingly, and in contrast to lymph node T cells, no perivascular T cells showed FoxP3 expression, a marker of regulatory T cells, at any stage of MS lesions. These observations suggest an enrichment of both IL-17+CD4+ and CD8+ T cells in active MS lesions as well as an important role for IL-17 in MS pathogenesis, with some remarkable differences from the experimental autoimmune encephalomyelitis model. Recent findings in the animal model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis, implicate a novel CD4+ T-cell subset (TH17), characterized by the secretion of interleukin-17 (IL-17), in disease pathogenesis. To elucidate its role in MS, brain tissues from patients with MS were compared to controls. We detected expression of IL-17 mRNA (by in situ hybridization) and protein (by immunohistochemistry) in perivascular lymphocytes as well as in astrocytes and oligodendrocytes located in the active areas of MS lesions. Further, we found a significant increase in the number of IL-17+ T cells in active rather than inactive areas of MS lesions. Specifically, double immunofluorescence showed that IL-17 immunoreactivity was detected in 79% of T cells in acute lesions, 73% in active areas of chronic active lesions, but in only 17% of those in inactive lesions and 7% in lymph node control tissue. CD8+, as well as CD4+, T cells were equally immunostained for IL-17 in MS tissues. Interestingly, and in contrast to lymph node T cells, no perivascular T cells showed FoxP3 expression, a marker of regulatory T cells, at any stage of MS lesions. These observations suggest an enrichment of both IL-17+CD4+ and CD8+ T cells in active MS lesions as well as an important role for IL-17 in MS pathogenesis, with some remarkable differences from the experimental autoimmune encephalomyelitis model. Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the central nervous system (CNS). Animal and human studies have shown that T cells and inflammatory cytokines play an important role in MS lesion pathogenesis.1Sospedra M Martin R Immunology of multiple sclerosis.Annu Rev Immunol. 2005; 23: 683-747Crossref PubMed Scopus (1830) Google Scholar Therefore, pathologically distinct areas in the brain can be characterized as acute, chronic active and inactive lesions, depending on the degree of mononuclear cell infiltrates and level of destruction of myelin sheaths. For many years, it was widely accepted that MS is a CD4+ T-helper 1 (TH1)-mediated disease.2Zamvil SS Steinman L The T lymphocyte in experimental allergic encephalomyelitis.Annu Rev Immunol. 1990; 8: 579-621Crossref PubMed Scopus (944) Google Scholar These TH1 cells are differentiated in response to the cytokine interleukin (IL)-12 and are themselves characterized by their expression of the proinflammatory cytokine interferon-γ. Most of this thinking originated from the results obtained from the animal model of MS, experimental autoimmune encephalomyelitis (EAE).2Zamvil SS Steinman L The T lymphocyte in experimental allergic encephalomyelitis.Annu Rev Immunol. 1990; 8: 579-621Crossref PubMed Scopus (944) Google Scholar However, these views were also first challenged in EAE by the finding that IL-12 knockout mice cannot generate TH1 cells, but are still susceptible to EAE, whereas IL-23 knockout mice are not.3Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3313) Google Scholar In addition to IL-6 and transforming growth factor-β, the cytokine IL-23 helps to expand a subpopulation of TH cells that specifically express the cytokines IL-17, IL-6, tumor necrosis factor-α,3Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3313) Google Scholar and IL-224Zheng Y Danilenko DM Valdez P Kasman I Eastham-Anderson J Wu J Ouyang W Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.Nature. 2007; 445: 648-651Crossref PubMed Scopus (1598) Google Scholar (TH17) and that have since been shown to play a crucial role in EAE.3Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3313) Google Scholar By contrast, naïve T cells receiving only transforming growth factor-β, IL-2, and retinoic acid signals differentiate into the distinct CD4+CD25+FoxP3+ regulatory T-cell population at the expense of TH17 cells.5Laurence A Tato CM Davidson TS Kanno Y Chen Z Yao Z Blank RB Meylan F Siegel R Hennighausen L Shevach EM O'Shea JJ Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation.Immunity. 2007; 26: 371-381Abstract Full Text Full Text PDF PubMed Scopus (1219) Google Scholar, 6Mucida D Park Y Kim G Turovskaya O Scott I Kronenberg M Cheroutre H Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid.Science. 2007; 317: 256-260Crossref PubMed Scopus (1653) Google Scholar When the TH17 cells were depleted from the T-cell population obtained from EAE mice, the remaining T cells no longer induced EAE after adoptive transfer.3Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3313) Google Scholar EAE severity was also greatly reduced on treatment with a monoclonal antibody to IL-173Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3313) Google Scholar; similarly, mice treated with an antibody against IL-23 failed to develop EAE.7Chen Y Langrish CL McKenzie B Joyce-Shaikh B Stumhofer JS McClanahan T Blumenschein W Churakovsa T Low J Presta L Hunter CA Kastelein RA Cua DJ Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis.J Clin Invest. 2006; 116: 1317-1326Crossref PubMed Scopus (503) Google Scholar Although EAE could still occur in IL-17 knockout mice, the disease was heavily attenuated,8Komiyama Y Nakae S Matsuki T Nambu A Ishigame H Kakuta S Sudo K Iwakura Y IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis.J Immunol. 2006; 177: 566-573PubMed Google Scholar and mice deficient for the key transcription factor for differentiation of TH17 cells, the orphan nuclear receptor RORγt, showed a mild EAE course and a delayed onset.9Ivanov II McKenzie BS Zhou L Tadokoro CE Lepelley A Lafaille JJ Cua DJ Littman DR The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (4100) Google Scholar However, although CD4+FoxP3+ regulatory T cells are present in the CNS during EAE, they are unable to contain the inflammatory damage.10Korn T Reddy J Gao W Bettelli E Awasthi A Petersen TR Backstrom BT Sobel RA Wucherpfennig KW Strom TB Oukka M Kuchroo VK Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation.Nat Med. 2007; 13: 423-431Crossref PubMed Scopus (687) Google Scholar In addition, IL-17 has been implicated not only in the animal model for MS, but also in various disease models for other autoimmune diseases, such as rheumatoid arthritis,11Nakae S Nambu A Sudo K Iwakura Y Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice.J Immunol. 2003; 171: 6173-6177PubMed Google Scholar inflammatory bowel disease,12Hue S Ahern P Buonocore S Kullberg MC Cua DJ McKenzie BS Powrie F Maloy KJ Interleukin-23 drives innate and T cell-mediated intestinal inflammation.J Exp Med. 2006; 203: 2473-2483Crossref PubMed Scopus (694) Google Scholar psoriasis,4Zheng Y Danilenko DM Valdez P Kasman I Eastham-Anderson J Wu J Ouyang W Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.Nature. 2007; 445: 648-651Crossref PubMed Scopus (1598) Google Scholar and uveitis.13Amadi-Obi A Yu CR Liu X Mahdi RM Clarke GL Nussenblatt RB Gery I Lee YS Egwuagu CE T(H)17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1.Nat Med. 2007; 13: 711-718Crossref PubMed Scopus (701) Google Scholar Although EAE and models for various other autoimmune diseases show a dominant role of TH17 cells in disease pathogenesis, few studies have addressed their role in their human disease prototypes. In fact, human TH17 cells seem to be distinctively different from those described in the mouse. Transforming growth factor-β is not needed for IL-17 production in human T cells and even inhibits IL-17 production, whereas IL-1 is a very effective and IL-6 a poor inducer of TH17 differentiation.14Acosta-Rodriguez EV Napolitani G Lanzavecchia A Sallusto F Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells.Nat Immunol. 2007; 8: 942-949Crossref PubMed Scopus (1546) Google Scholar, 15Wilson NJ Boniface K Chan JR McKenzie BS Blumenschein WM Mattson JD Basham B Smith K Chen T Morel F Lecron JC Kastelein RA Cua DJ McClanahan TK Bowman EP de Waal Malefyt R Development, cytokine profile and function of human interleukin 17-producing helper T cells.Nat Immunol. 2007; 8: 950-957Crossref PubMed Scopus (1681) Google Scholar The role of human IL-23 in inducing IL-17-secreting cells is, however, unclear, because it has been shown to be either an ineffective inducer14Acosta-Rodriguez EV Napolitani G Lanzavecchia A Sallusto F Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells.Nat Immunol. 2007; 8: 942-949Crossref PubMed Scopus (1546) Google Scholar or a highly potent inducer15Wilson NJ Boniface K Chan JR McKenzie BS Blumenschein WM Mattson JD Basham B Smith K Chen T Morel F Lecron JC Kastelein RA Cua DJ McClanahan TK Bowman EP de Waal Malefyt R Development, cytokine profile and function of human interleukin 17-producing helper T cells.Nat Immunol. 2007; 8: 950-957Crossref PubMed Scopus (1681) Google Scholar of TH17 cells. IL-17-secreting cells have been detected in MS16Matusevicius D Kivisakk P He B Kostulas N Ozenci V Fredrikson S Link H Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.Mult Scler. 1999; 5: 101-104PubMed Google Scholar and also in rheumatoid arthritis17Kotake S Udagawa N Takahashi N Matsuzaki K Itoh K Ishiyama S Saito S Inoue K Kamatani N Gillespie MT Martin TJ Suda T IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis.J Clin Invest. 1999; 103: 1345-1352Crossref PubMed Scopus (1412) Google Scholar, 18Hwang SY Kim HY Expression of IL-17 homologs and their receptors in the synovial cells of rheumatoid arthritis patients.Mol Cells. 2005; 19: 180-184PubMed Google Scholar and inflammatory bowel disease.19Nielsen OH Kirman I Rudiger N Hendel J Vainer B Upregulation of interleukin-12 and -17 in active inflammatory bowel disease.Scand J Gastroenterol. 2003; 38: 180-185Crossref PubMed Scopus (213) Google Scholar In MS increased numbers of mononuclear cells have been shown to express IL-17 mRNA in the peripheral blood, particularly during exacerbations, with a higher proportion of IL-17 mRNA-expressing cells in the cerebrospinal fluid.16Matusevicius D Kivisakk P He B Kostulas N Ozenci V Fredrikson S Link H Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.Mult Scler. 1999; 5: 101-104PubMed Google Scholar They have also recently been shown to migrate preferentially across a model for the blood-brain barrier.20Kebir H Kreymborg K Ifergan I Dodelet-Devillers A Cayrol R Bernard M Giuliani F Arbour N Becher B Prat A Human T(H)17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation.Nat Med. 2007; 13: 1173-1175Crossref PubMed Scopus (1276) Google Scholar In addition, IL-17 levels seem to be elevated in cerebrospinal fluid samples from MS patients, in particular in opticospinal MS patients.21Ishizu T Osoegawa M Mei FJ Kikuchi H Tanaka M Takakura Y Minohara M Murai H Mihara F Taniwaki T Kira J Intrathecal activation of the IL-17/IL-8 axis in opticospinal multiple sclerosis.Brain. 2005; 128: 988-1002Crossref PubMed Scopus (301) Google Scholar Furthermore, early microarray analyses of different MS lesions noted increased levels of IL-17 mRNA transcripts, particularly in more chronic lesions,22Lock C Hermans G Pedotti R Brendolan A Schadt E Garren H Langer-Gould A Strober S Cannella B Allard J Klonowski P Austin A Lad N Kaminski N Galli SJ Oksenberg JR Raine CS Heller R Steinman L Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis.Nat Med. 2002; 8: 500-508Crossref PubMed Scopus (1443) Google Scholar along with other genes for proinflammatory cytokines. However, it has not yet been investigated whether IL-17-producing cells actually invade into the CNS in MS patients and can be found in the inflammatory infiltrates. Here, we address this question for the first time, identifying the different cell types in MS brains that express and produce IL-17. We show that mRNA expression and protein production of IL-17 is primarily restricted to the active areas of MS lesions. Not only T cells but also, surprisingly, astrocytes and oligodendrocytes produce the cytokine in inflammatory areas of different lesions. In the active areas of acute lesions and the active borders of chronic active lesions, we found higher densities of IL-17+ T cells than in inactive areas, normal-appearing white matter (NAWM), and control tissue. We also demonstrate high proportions of CD8+ as well as CD4+ T cells that are IL-17+ with a similar distribution. By contrast, we could not detect any FoxP3+ regulatory T cells in any MS brain tissues, suggesting an absence of T-cell suppression in the CNS. The predominance of both CD4+IL-17+ and CD8+IL-17+ T cells within active areas, together with the known evidence of IL-17 as a proinflammatory cytokine, supports a pathogenic role for IL-17 in MS. All brain tissues were obtained from the UK Multiple Sclerosis Tissue Bank, the Thomas Willis Oxford Brain Collection, or NeuroResource, University College London Institute of Neurology. Twenty-two frozen tissue blocks were used in this study, comprising five acute, six chronic active, and five inactive lesions, three tissue samples of NAWM, and four control (normal) brains. These were obtained from 14 MS patients and 4 patients with nonneurological diseases (Table 1). To characterize the lesions, oil red O (ORO), Luxol fast blue-cresyl violet, and hematoxylin and eosin stainings were performed to identify the centers and borders of the lesions, lesion activity, the hypercellularity (inflammatory cell infiltrate), and areas of hypocellularity (loss of parenchymal cells in inactive lesions). Lesions were characterized as follows: 1) active lesions by hypercellularity because of lymphocytic infiltration and high number of macrophages highly stained with ORO because of the positive lipid droplets from the breakdown of myelin; 2) chronic active lesions by hypercellular active border stained highly positive with ORO and by a hypocellular demyelinated center with a very low density of macrophages, ORO-negative; 3) inactive lesions by demyelination and a high degree of hypocellularity because of the significant loss of parenchymal cells and the limited number of inflammatory infiltrates stained negative for ORO. In all MS cases, the clinical diagnosis of MS had been made during life and confirmed by neuropathological autopsy examination. This study was approved by the Oxford Regional NHS Research Ethics Committee, UK.Table 1Cryostat MS LesionsCaseBlockSample typeDiseaseAge (years)Disease duration (years)MS typeCause of death1AcuteMS298Secondary progressiveBronchopneumonia2AAcuteMS5416Primary progressiveMyocardial infarct3AAcuteMS409Secondary progressiveBronchopneumonia4AAcuteMSNANANANA4BAcuteMSNANANANA5Chronic activeMS6525Secondary progressiveBronchopneumonia6Chronic activeMS4622Secondary progressiveBronchopneumonia7Chronic activeMS5920Secondary progressiveBronchopneumonia8Chronic activeMS5110Secondary progressiveBronchopneumonia2BChronic activeMS5416Primary progressiveMyocardial infarct2CChronic activeMS5416Primary progressiveMyocardial infarct9InactiveMS4622Secondary progressiveBronchopneumonia10InactiveMS6729Secondary progressiveBronchopneumonia11InactiveMS7132Secondary progressiveBronchopneumonia2DInactiveMS5416Primary progressiveMyocardial infarct12InactiveMSNANANANA13NAWMMS426Primary progressiveBronchopneumonia3BNAWMMS409Secondary progressiveBronchopneumonia14NAWMMS39NANAPulmonary embolism, pneumonia15Control75CVA, aspiration pneumonia16Control35Carcinoma of the tongue17Control73Aortic aneurysm18Control49Myocardial infarctNAWM, normal appearing white matter; NA, not available. Open table in a new tab NAWM, normal appearing white matter; NA, not available. For the generation of the riboprobes, a 451-base IL-17 cDNA fragment was obtained by polymerase chain reaction from an IL-17 cDNA inserted in pDNR-dual donor vector (GenBank accession number NM_002190; Bio Techniques, Westborough, MA) and verified by sequencing. Digoxygenin-labeled sense and anti-sense riboprobes recognizing IL-17 nucleotide sequences 3 to 453 were prepared by in vitro transcription. Sections were processed for in situ hybridization cytochemistry as previously described23Craner MJ Kataoka Y Lo AC Black JA Baker D Waxman SG Temporal course of upregulation of Na (v) 1.8 in Purkinje neurons parallels the progression of clinical deficit in experimental allergic encephalomyelitis.J Neuropathol Exp Neurol. 2003; 62: 968-975PubMed Google Scholar using an isoform-specific riboprobe to IL-17. In brief, tissue sections were cut at 10 μm and fixed for 11 minutes with 4% paraformaldehyde followed by treatment with proteinase K (Sigma-Aldrich, Dorset, UK) for 20 minutes at room temperature under RNase-free conditions. Hybridization with the digoxigenin-labeled riboprobe (DIG RNA labeling kit; Roche Diagnostics, Mannheim, Germany) was performed by overnight incubation at 68°C. After washes, the sections were incubated with anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (1:2000) (Roche Diagnostics) overnight at 4°C and visualized using nitro blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyl phosphate p-toluidine salt (Roche Diagnostics) chromogenic reaction. Positive controls of fresh frozen tonsil demonstrated specific signal in cells within the parafollicular region (T cells) (data not shown). Sense riboprobes yielded no signals in in situ hybridization (see Figure 1). Frozen sections (10 μm thick) were air-dried, fixed in 0.4% paraformaldehyde, and quenched in ammonium chloride (0.1 mmol/L). Endogenous peroxidase was blocked by incubation with 1 to 3% hydrogen peroxide for 45 minutes. Consecutive sections were stained with mouse anti-CD3 1/100 dilution (DAKO, Cambridge, UK) and mouse anti-IL-17 1/100 dilution (R&D Systems, Abingdon, UK) antibodies overnight at 4°C. Slides were developed with the Envision kit by incubating for 1 hour at room temperature with the horseradish peroxidase anti-rabbit/mouse complex (DAKO). Staining was performed using diaminobenzidine (DAKO), as chromogenic substrate for up to 5 minutes. We used double immunofluorescence to localize IL-17+ cells. Tissue sections were fixed as described above and then incubated with anti-IL-17 antibody (R&D Systems), 1/100 dilution overnight at 4°C, followed by staining using the tyramide signal amplification kit with Alexa Fluor 488 anti-mouse Ig (Invitrogen, Paisley, UK). After washing, slides were then incubated overnight with a second antibody: rat anti-CD3, 1/100 dilution (Serotec, Oxford, UK), rat anti-CD4, 1/100 dilution (MCA4b4G, Serotec), rat anti-CD8, 1/100 dilution (YTC 182.20, Serotec), rabbit anti-GFAP, 1/1000 dilution (DAKO), sheep anti-CAII (carbonic anhydrase isoenzyme II) 1/300 dilution (Serotec), rat anti-MHC class II, 1/100 dilution (Serotec), or rabbit anti-FoxP3 1/200 dilution (ab10563; Abcam, Cambridge, UK). Appropriate secondary antibodies included Cy3 anti-rat Ig, 1/100 dilution (Jackson Laboratory, Suffolk, UK), and Alexa Fluor 568 anti-rabbit 1/200 or anti-sheep 1/200 Ig (Invitrogen) for 4 hours. As negative controls for each second layer, the same procedure as above was followed except that the primary antibodies were omitted. No staining was seen in these sections. Positive control sections for IL-17 consisted of fresh frozen tonsillar tissue. Areas of different activity were used for quantification, from acute, chronic active, and inactive lesions as well as NAWM, control brains, and lymphatic tissues. In active areas, hypercellular (oil red-positive) areas, or in inactive areas, hypocellular (oil red-negative) areas >900 μm away from lesion borders were quantified. Digital images of tissue sections were captured using an Olympus light microscope BX41 and a Zeiss camera (Zeiss, Thornwood, NY). To calculate the density of IL-17+ and CD3+ cells in areas of different activity, consecutive sections of all MS cases and controls were stained using diaminobenzidine immunohistochemistry. Multiple images of different perivascular cuffs were acquired and the areas calculated by the Axion Image software (Zeiss). Within the cuffs, our double-immunofluorescence data demonstrate that only cells with T-cell morphology label for IL-17. The numbers of CD3+ (T-cell marker) and IL-17+ cells with such morphology were counted by eye within the predefined area of each perivascular cuff and the density expressed as cells/mm2. In each lesion, three to six cuffs with a total area >2 × 105 μm2 were counted. A second blinded observer counted the number of CD3+ and IL-17+ cells from a representative number of lesions of various activities, differences being <4% between observers. To investigate IL-17 protein co-localization with CD4+ and CD8+ T cells, multiple images were acquired using a confocal microscope (Bio-Rad, Hercules, CA). For counting percentages of CD3+, CD4+, and CD8+ cells in lesion areas of different activity, multiple images were taken from all of the cuffs, and their cells counted using ImageJ software (National Institutes of Health, Bethesda, MD). Statistical significance was calculated using Student's t-test for unequal variances and analysis of variance with multiple comparisons as appropriate. Data are presented as mean ± SEM. *P < 0.05 denotes statistically significant differences. To assess potential contributions of IL-17 in the pathogenesis of MS, we compared the expression of its mRNA and protein in acute, chronic active, and inactive MS lesions and in NAWM and control brain tissue. Using a specific anti-sense riboprobe, we detected increased expression of IL-17 mRNA within active areas of acute lesions and in the borders of chronic active lesions relative to inactive areas of chronic active lesions (Figure 1A) and NAWM. Interestingly, the morphological characteristics and relative distribution of the cells suggested that IL-17 mRNA was expressed in T cells (Figure 1B), astrocytes (Figure 1C), and oligodendrocytes24Morris CS Esiri MM Sprinkle TJ Gregson N Oligodendrocyte reactions and cell proliferation markers in human demyelinating diseases.Neuropathol Appl Neurobiol. 1994; 20: 272-281Crossref PubMed Scopus (40) Google Scholar (Figure 1D). Moreover, within all lesions examined, there was a consistently higher level of IL-17 expression within all cell types identified in active areas of MS lesions (acute and chronic active lesion borders) than in inactive areas or control tissue (Figure 1A and data not shown). To investigate the production of IL-17 protein in immune and glial cells, we used double immunofluorescence. We found that, within acute or active borders of chronic active lesions, T cells (CD3+; Figure 2, A–C), astrocytes (GFAP+; Figure 2, G–I), and oligodendrocytes (CAII+; Figure 2, J–L) were also IL-17-positive. In contrast, microglia/macrophages (MHCII+; Figure 2, D–F) showed no detectable immunoreactivity for IL-17. In control CNS tissue, astrocytes were only marginally IL-17+ (Supplementary Figure 1A, see http://ajp.amjpathol.org), whereas their activated counterparts in NAWM from MS patients showed greater IL-17 immunoreactivity than control brains (data not shown); they were compared to robust IL-17 immunostaining in acute lesions and at chronic active lesion borders (Supplementary Figure 1, B and C; see http://ajp.amjpathol.org). In inactive lesions, no clear staining of astrocyte cell bodies was evident, but the network of astrocytic processes stained positively for IL-17 (Supplementary Figure 1D, see http://ajp.amjpathol.org). Because recent studies in animal models suggest an important role for the highly proinflammatory TH17 cells in the development of autoimmunity, we next focused on their distribution within MS lesions. As in previous studies,25Nyland H Mork S Matre R In-situ characterization of mononuclear cell infiltrates in lesions of multiple sclerosis.Neuropathol Appl Neurobiol. 1982; 8: 403-411Crossref PubMed Scopus (35) Google Scholar, 26Traugott U Reinherz EL Raine CS Multiple sclerosis: distribution of T cell subsets within active chronic lesions.Science. 1983; 219: 308-310Crossref PubMed Scopus (344) Google Scholar, 27Hauser SL Bhan AK Gilles F Kemp M Kerr C Weiner HL Immunohistochemical analysis of the cellular infiltrate in multiple sclerosis lesions.Ann Neurol. 1986; 19: 578-587Crossref PubMed Scopus (318) Google Scholar, 28Woodroofe MN Bellamy AS Feldmann M Davison AN Cuzner ML Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth.J Neurol Sci. 1986; 74: 135-152Abstract Full Text PDF PubMed Scopus (183) Google Scholar we found significantly higher densities of T cells especially within perivascular areas of acute lesions (Figure 3A) and active borders of chronic active lesions (Figure 3C) (2631 ± 521 cells/mm2), but also within inactive lesions (Figure 3G) and inactive areas of chronic active lesions (Figure 3E) (1080 ± 208 cells/mm2), than in NAWM (Figure 3I) and control tissue (Figure 3K) (495 ± 143 cells/mm2, P = 0.01; summarized in Figure 4A).Figure 4Density of IL-17+ T cells in MS lesions of different activity. Quantitative analysis of perivascular CD3+ (A) and IL-17+ (B) T-cell density in areas of MS lesions with different activity, in NAWM, and in control brain. Histograms demonstrate a significantly higher density of T cells (A) and IL-17+ T cells (B) within perivascular active areas (acute lesions/active border of chronic active lesions) than in inactive lesions/inactive areas of chronic active lesions or NAWM and control tissue (n = 4). In contrast, there is no significant difference in the density of IL-17+ T cells within inactive areas and control tissue. The proportion of T cells that are IL-17+ was significantly higher in active than inactive areas of chronic active lesions (P = 0.0005).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Consistent with this observation, we noted significantly higher densities of IL-17+ T cells within acute lesions (Figure 3B) and active borders of chronic active lesions (Figure 3D) (1687 ± 284 cells/mm2) than in NAWM (Figure 3J) and control tissue (Figure 3L) (333 ± 105 cells/mm2, P = 0.001). However, IL-17+ T cells were nearly 10× less frequent within inactive lesions (Figure 3H) and inactive regions of chronic active lesions (Figure 3F) (198 ± 92 cells/mm2) and in controls (Figure 3L) (P = 0.2). Again these analyses are summarized in Figure 4B. Interestingly, there was a significantly higher proportion of IL-17+ T cells in acute lesions and active regions of chronic active lesions (71.2 ± 5%) than in inactive lesions and inactive areas of chronic active lesions (24 ± 9%, P = 0.0005). Examination of NAWM and control brain tissue de
